Our long term goal is to develop a more accurate test that can preoperatively distinguish a benign from a malignant thyroid nodule. Currently, fine-needle aspiration (FNA) cytology is the best non-surgical diagnostic tool for evaluating a thyroid nodule. However, approximately 30% of all nodules are classified as suspicious. Because the current method is based on morphological features, and fails to improve the sensitivity and specificity, it remains a frequent clinical problem with challenges in particular to the pathologist. To address this problem, we previously performed gene expression profiling both a follicular thyroid adenoma (FTA), and a follicular thyroid carcinoma (FTC). This profiling and subsequent analysis revealed that four novel markers (DDIT3, ARG2, C1orf24 and ITM1) differed between the two tumors and a linear combination of expression levels distinguished FTC from FTA with an estimated predictive accuracy of 0.83. Commercially available antibodies for DDIT3 and ARG2 were used for further validation in an independent set of FTA and FTC paraffin-embedded sections. Sensitivity and specificity were 85% and 91% respectively for each antibody. Using the two antibodies in combination did not improve the estimates. We custom produced antibodies for C1or24 and ITM1 and tested them in the aforementioned set of FTA and FTC sections. We achieved a sensitivity of 100%. Furthermore, these novel markers can reliably classify other thyroid lesions into benign or malignant classes. Our hypothesis is that gene expression profiling will locate novel diagnostic markers that can improve the specificity of the test. We will use Serial Analysis of Gene Expression (SAGE), to profile Hurthle cell adenoma which was originally misclassified as malignant and, therefore, predicted to improve the specificity of preoperative diagnosis test. Our next goal is to develop a test that can be routinely used as an adjuvant with FNA cytology. To achieve this goal, we propose in R33 phase of this application to evaluate the effectiveness of both antibody and quantitative RT-PCR tests to diagnose thyroid nodules. To assess the accuracy and feasibility of both tests, the results from these analyses will be compared with final histology. The development of an innovative and more accurate preoperative diagnostic test for evaluating thyroid nodules will not only result in progress in cancer diagnosis but will also improve treatment decisions while reducing long-term health costs.